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Fulltext Detection of Strongyloides stercoralis infection among cancer patients in a major hospital in Kelantan, Malaysia

Zueter AM, Mohamed Z, Abdullah AD, Mohamad N, Arifin N, Othman N, et al.

Singapore Med J, 2014 Jul;55(7):367-71.
PMID: 25091885

INTRODUCTION: Strongyloidiasis is one of the most commonly neglected but clinically important parasitic infections worldwide, especially among immunocompromised patients. Evidence of infection among immunocompromised patients in Malaysia is, however, lacking. In this study, microscopy, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs) were used to detect Strongyloides stercoralis (S. stercoralis) infection among cancer patients in a Malaysian hospital.

METHODS: A total of 192 stool and serum samples were collected from cancer patients who were receiving chemotherapy with or without steroid treatment at a hospital in northeastern Malaysia. Stool samples were examined for S. stercoralis using parasitological methods and real-time PCR. Serology by ELISA was performed to detect parasite-specific immunoglobulin G (IgG), IgG4 and immunoglobulin E (IgE) antibodies. For comparison, IgG4- and IgG-ELISAs were also performed on the sera of 150 healthy individuals from the same area.

RESULTS: Of the 192 samples examined, 1 (0.5%) sample was positive for S. stercoralis by microscopy, 3 (1.6%) by real-time PCR, 8 (4.2%) by IgG-ELISA, 6 (3.1%) by IgG4-ELISA, and none was positive by IgE-ELISA. In comparison, healthy blood donors had significantly lower prevalence of parasite-specific IgG (2.67%, p < 0.05) and IgG4 (2.67%, p < 0.05) responses.

CONCLUSION: This study showed that laboratory testing may be considered as a diagnostic investigation for S. stercoralis among immunocompromised cancer patients.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Detection of Tuberculosis (TB) using gold standard method, direct sputum smears microscopy, PCR, qPCR and electrochemical DNA sensor: a mini review

Che Engku Noramalina Che-Engku-Chik, Siti Sarah Othman, Helmi Wasoh, Nor Azah Yusof, Jaafar Abdullah, Mohd Hazani Mat Zaid

Journal of Biochemistry, Microbiology and Biotechnology, 2016;4(2):16-21.
MyJurnal

Despite the continued effort globally made to control the growing case of Tuberculosis (TB), it
continues to be regarded as the second deadliest disease after the HIV. There are various
methods developed to diagnose TB, most of which having the criteria of sensitive, selective,
cheap and portable to be used in robust applications. Even with the advancement in medication,
the important keys including early stage diagnosis is yet to be considered. In diagnosing TB, the
only technique remained as the gold standard method is the culturing method, which is the Acid
Fast Bacilli (AFB) staining. On the other hand, molecular technique utilising Polymerase Chain
Reaction (PCR) assay is preferred as a non-culturing method. Additionally, as molecular
techniques become advanced, real-time PCR or quantitative PCR (qPCR) using multiple probes
in one shot has raised interest among researchers, because it can skip the process of gel
electrophoresis. Recently, researchers have been working on electrochemical DNA sensors
which are sensitive, selective, rapid, cheap and can meet with point of care (POC) testing
requirements to diagnose TB.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Detection of human herpesvirus 6 (HHV-6) in saliva of healthy adults in Malaysia

Choo, H.L., Shoji, Y., Leong, C.O.

Malaysian Journal of Medicine and Health Sciences, 2012;8(2):55-64.
MyJurnal

Human herpesvirus-6 (HHV-6) levels have been considered as markers for various diseases. The aim of this study was to evaluate the prevalence of HHV-6 infection in healthy adults in Malaysia. Methods: The level of HHV-6 in saliva was investigated in 36 healthy adults, age 19 to 23 years, at Kuala Lumpur, Malaysia using variant-specific TaqmanTM quantitative real-time PCR (qPCR). Results: The amount of HHV-6 DNA in the saliva of healthy adults ranged from negative to 10,000 HHV-6 genomes/ml of saliva (median, 360 genomes/ml of saliva). Of the 36 samples tested, 30 (83%) contained HHV-6 DNA. HHV-6B was the only variant detected in the saliva of all the positive cases. Conclusions: The detection of HHV-6 DNA in saliva by real-time PCR assay provides a sensitive and specific quantitation of HHV-6. Our pilot study suggests the wide prevalence of HHV-6 in saliva from
healthy adults.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR

Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R

Trop Biomed, 2012 Sep;29(3):434-42.
PMID: 23018507

Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample

Saidin S, Yunus MH, Othman N, Lim YA, Mohamed Z, Zakaria NZ, et al.

Pathog Glob Health, 2017 May;111(3):128-136.
PMID: 28335696 DOI: 10.1080/20477724.2017.1300421

Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml-1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml-1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Development and validation of pan-Leptospira Taqman qPCR for the detection of Leptospira spp. in clinical specimens

Mohd Ali MR, Mohd Safee AW, Ismail NH, Abu Sapian R, Mat Hussin H, Ismail N, et al.

Mol Cell Probes, 2018 04;38:1-6.
PMID: 29524642 DOI: 10.1016/j.mcp.2018.03.001

BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely.
METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients.
RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri.
CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

Chua KH, Lim SC, Ng CC, Lee PC, Lim YA, Lau TP, et al.

Sci Rep, 2015;5:15671.
PMID: 26507008 DOI: 10.1038/srep15671

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNex(TM), a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1-100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNex(TM). This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNex(TM). The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of TaqMan-based real-time RT-PCR assay based on N gene for the quantitative detection of feline morbillivirus

Makhtar ST, Tan SW, Nasruddin NA, Abdul Aziz NA, Omar AR, Mustaffa-Kamal F

BMC Vet Res, 2021 Mar 23;17(1):128.
PMID: 33757494 DOI: 10.1186/s12917-021-02837-6

BACKGROUND: Morbilliviruses are categorized under the family of Paramyxoviridae and have been associated with severe diseases, such as Peste des petits ruminants, canine distemper and measles with evidence of high morbidity and/or could cause major economic loss in production of livestock animals, such as goats and sheep. Feline morbillivirus (FeMV) is one of the members of Morbilliviruses that has been speculated to cause chronic kidney disease in cats even though a definite relationship is still unclear. To date, FeMV has been detected in several continents, such as Asia (Japan, China, Thailand, Malaysia), Europe (Italy, German, Turkey), Africa (South Africa), and South and North America (Brazil, Unites States). This study aims to develop a TaqMan real-time RT-PCR (qRT-PCR) assay targeting the N gene of FeMV in clinical samples to detect early phase of FeMV infection.
RESULTS: A specific assay was developed, since no amplification was observed in viral strains from the same family of Paramyxoviridae, such as canine distemper virus (CDV), Newcastle disease virus (NDV), and measles virus (MeV), and other feline viruses, such as feline coronavirus (FCoV) and feline leukemia virus (FeLV). The lower detection limit of the assay was 1.74 × 104 copies/μL with Cq value of 34.32 ± 0.5 based on the cRNA copy number. The coefficient of variations (CV) values calculated for both intra- and inter-assay were low, ranging from 0.34-0.53% and 1.38-2.03%, respectively. In addition, the clinical sample evaluation using this assay showed a higher detection rate, with 25 (35.2%) clinical samples being FeMV-positive compared to 11 (15.5%) using conventional RT-PCR, proving a more sensitive assay compared to the conventional RT-PCR.
CONCLUSIONS: The TaqMan-based real-time RT-PCR assay targeting the N gene described in this study is more sensitive, specific, rapid, and reproducible compared to the conventional RT-PCR assay targeting the N gene, which could be used to detect early infection in cats.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods; Real-Time Polymerase Chain Reaction/veterinary*
Fulltext Development of a SYBR green based real-time polymerase chain reaction assay for specific detection and quantification of Vibrio parahaemolyticus from food and environmental samples

Micky, V., Nur Quraitu’ Aini, T., Velnetti, L., Patricia Rowena, M.B., Christy, C., Lesley Maurice, B.

International Food Research Journal, 2014;21(3):921-927.
MyJurnal

Vibrio parahaemolyticus is a foodborne pathogen and their human infection is regularly associated with the consumption of raw or undercooked seafood and contaminated water supplies. Many conventional biochemical identification and confirmation procedures are performed to detect the presence of this pathogen, both from seafood or environmental samples. However, these procedures not only require two or more days to complete, they do not have the capabilities to determine the number of V. parahaemolyticus cells in any given samples. Thus, in this study we describe the development of a rapid SYBR green based real-time PCR assay, targeting the thermo labile (tl) gene of V. parahaemolyticus for the detection and enumeration of this bacterium from seafood and environmental samples. We report that the real-time PCR assay and the primers designed are highly specific, and only generated the desired amplicons with V. parahaemolyticus DNA samples against other bacteria and fungi species. Our assay is also highly sensitive, and, is able to detect V. parahaemolyticus with high coefficient values in concentrations as low as 1.0 pg/μl DNA for pure genomic DNA solutions and 10 cells/ml in serially diluted cell suspension and spiked samples. This assay can be completed in less than 3 hours and may be used as a tool for rapid determination of V. parahaemolyticus densities in the food industries, environmental risk assessment and for clinical diagnostics purposes.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Development of a TaqMan minor groove binding probe-based quantitative reverse transcription polymerase chain reaction for the detection and quantification of Zika virus

Chin KL, Teoh BT, Sam SS, Loong SK, Tan KK, Azizan NS, et al.

Trop Biomed, 2022 Dec 01;39(4):518-523.
PMID: 36602210 DOI: 10.47665/tb.39.4.005

Zika virus (ZIKV) infection has emerged as a global health concern following epidemic outbreaks of severe neurological disorders reported in Pacific and Americas since 2016. Therefore, a rapid, sensitive and specific diagnostic test for ZIKV infection is critical for the appropriate patient management and the control of disease spread. A TaqMan minor groove binding (MGB) probe-based quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the conserved sequence regions of 463 ZIKV NS2B genes. The designed ZIKV qRT-PCR assay was evaluated for its detection limit, strain coverage and cross-reactivity. We further assessed the clinical applicability of qRT-PCR assay for ZIKV RNA detection using a total 18 simulated clinical specimens. The detection limit of the qRT-PCR assay was 11.276 ZIKV RNA copies at the 95% probability level (probit analysis, p<= 0.05). Both Asian and African ZIKV strains were detected by the qRT-PCR assay without cross-reacting with DENV-1, DENV-2, DENV-3, DENV-4, CHIKV, JEV, LGTV, GETV and SINV. The qRT-PCR assay demonstrated a perfect agreement (k = 1.000, P < 0.001) with the reference assay; the sensitivity and specificity of the qRT-PCR assay were 100% (95% CI= 79.6-100) and 100% (95% CI= 43.9-100) respectively. The qRT-PCR assay developed in this study is a useful diagnostic tool for the broad coverage detection and quantification of both the Asian and African ZIKV strains.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of a cartilage composite utilizing porous tantalum, fibrin, and rabbit chondrocytes for treatment of cartilage defect

Jamil K, Chua KH, Joudi S, Ng SL, Yahaya NH

J Orthop Surg Res, 2015;10:27.
PMID: 25889942 DOI: 10.1186/s13018-015-0166-z

Functional tissue engineering has emerged as a potential means for treatment of cartilage defect. Development of a stable cartilage composite is considered to be a good option. The aim of the study was to observe whether the incorporation of cultured chondrocytes on porous tantalum utilizing fibrin as a cell carrier would promote cartilage tissue formation.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of a reverse transcription recombinase polymerase amplification assay for rapid and direct visual detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)

Lau YL, Ismail IB, Mustapa NIB, Lai MY, Tuan Soh TS, Haji Hassan A, et al.

PLoS One, 2021;16(1):e0245164.
PMID: 33406112 DOI: 10.1371/journal.pone.0245164

Rapid diagnosis is an important intervention in managing the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak. Real time reverse transcription polymerase chain reaction (RT-qPCR) remains the primary means for diagnosing the new virus strain but it is time consuming and costly. Recombinase polymerase amplification (RPA) is an isothermal amplification assay that does not require a PCR machine. It is an affordable, rapid, and simple assay. In this study, we developed and optimized a sensitive reverse transcription (RT)-RPA assay for the rapid detection of SARS-CoV-2 using SYBR Green I and/or lateral flow (LF) strip. The analytical sensitivity and specificity of the RT-RPA assay were tested by using 10-fold serial diluted synthetic RNA and genomic RNA of similar viruses, respectively. Clinical sensitivity and specificity of the RT-RPA assay were carried out using 78 positive and 35 negative nasopharyngeal samples. The detection limit of both RPA and RT-qPCR assays was 7.659 and 5 copies/μL RNA, respectively with no cross reactivity with other viruses. The clinical sensitivity and specificity of RT-RPA were 98% and 100%, respectively. Our study showed that RT-RPA represents a viable alternative to RT-qPCR for the detection of SARS-CoV-2, especially in areas with limited infrastructure.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Fulltext Development of conventional and real-time multiplex PCR assays for the detection of nosocomial pathogens

Anbazhagan D, Mui WS, Mansor M, Yan GO, Yusof MY, Sekaran SD

Braz J Microbiol, 2011 Apr;42(2):448-58.
PMID: 24031653 DOI: 10.1590/S1517-83822011000200006

Nosocomial infections are major clinical threats to hospitalised patients and represent an important source of morbidity and mortality. It is necessary to develop rapid detection assays of nosocomial pathogens for better prognosis and initiation of antimicrobial therapy in patients. In this study, we present the development of molecular methods for the detection of six common nosocomial pathogens including Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. Conventional multiplex PCR and SYBR Green based real time PCR assays were performed using genus and species specific primers. Blind testing with 300 clinical samples was also carried out. The two assays were found to be sensitive and specific. Eubacterial PCR assay exhibited positive results for 46 clinical isolates from which 43 samples were detected by real time PCR assay. The sensitivity of the assay is about 93.7% in blind test isolates. The PCR results were reconfirmed using the conventional culture method. This assay has the potential to be a rapid, accurate and highly sensitive molecular diagnostic tool for simultaneous detection of Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. This assay has the potential to detect nosocomial pathogens within 5 to 6 hours, helping to initiate infection control measures and appropriate treatment in paediatric and elderly (old aged) patients, pre-and post surgery patients and organ transplant patients and thus reduces their hospitalization duration.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of conventional and real-time reverse transcription polymerase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes

Petz LN, Turell MJ, Padilla S, Long LS, Reinbold-Wasson DD, Smith DR, et al.

Am J Trop Med Hyg, 2014 Oct;91(4):666-71.
PMID: 25114013 DOI: 10.4269/ajtmh.13-0218

Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Development of multiplex real-time PCR for the rapid detection of five bacterial causes of community acquired pneumonia

Al-Marzooq F, Imad MA, How SH, Kuan YC

Trop Biomed, 2011 Dec;28(3):545-56.
PMID: 22433883 MyJurnal

Establishing a microbial diagnosis for patients with community-acquired pneumonia (CAP) is still challenging and is often achieved in only 30-50% of cases. Polymerase chain reaction (PCR) has been shown to be more sensitive than conventional microbiological methods and it could help to increase the microbial yield for CAP patients. This study was designed to develop, optimize and evaluate multiplex real-time PCR as a method for rapid differential detection of five bacterial causes of CAP namely Streptococcus pneumoniae, Burkholderia pseudomallei and atypical bacterial pathogens, Mycoplasma pneumoniae, Chlamydophila pneumoniae and Legionella pneumophila. Duplex and triplex real-time PCR assays were developed using five sets of primers and probes that were designed based on an appropriate specific gene for each of the above CAP pathogens. The performance of primers for each organism was tested using SYBR Green melt curve analysis following monoplex realtime PCR amplification. Monoplex real-time PCR assays were also used to optimize each primers-probe set before combining them in multiplex assays. Two multiplex real-time PCR assays were then optimized; duplex assay for the differential detection of S. pneumoniae and B. pseudomallei, and triplex assay for the atypical bacterial pathogens. Both duplex and triplex real-time PCR assays were tested for specificity by using DNA extracted from 26 related microorganisms and sensitivity by running serial dilutions of positive control DNAs. The developed multiplex real-time PCR assays shall be used later for directly identifying CAP causative agents in clinical samples.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Fulltext Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples

Rahumatullah A, Khoo BY, Noordin R

Trop Biomed, 2015 Jun;32(2):376-85.
PMID: 26691266 MyJurnal

Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods*
Fulltext Diagnostic Potential of an IgE-ELISA in Detecting Strongyloidiasis

Ahmad H, Balachandra D, Arifin N, Nolan TJ, Lok JB, Hayat Khan A, et al.

Am J Trop Med Hyg, 2020 12;103(6):2288-2293.
PMID: 32996454 DOI: 10.4269/ajtmh.20-0265

Strongyloides stercoralis infection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate. However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial Strongyloides IgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate. Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11). Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n = 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative. The optical density values of group IB sera were also significantly lower than those of group IA (P < 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Diagnostic tools in childhood malaria

Amir A, Cheong FW, De Silva JR, Lau YL

Parasit Vectors, 2018 01 23;11(1):53.
PMID: 29361963 DOI: 10.1186/s13071-018-2617-y

Every year, millions of people are burdened with malaria. An estimated 429,000 casualties were reported in 2015, with the majority made up of children under five years old. Early and accurate diagnosis of malaria is of paramount importance to ensure appropriate administration of treatment. This minimizes the risk of parasite resistance development, reduces drug wastage and unnecessary adverse reaction to antimalarial drugs. Malaria diagnostic tools have expanded beyond the conventional microscopic examination of Giemsa-stained blood films. Contemporary and innovative techniques have emerged, mainly the rapid diagnostic tests (RDT) and other molecular diagnostic methods such as PCR, qPCR and loop-mediated isothermal amplification (LAMP). Even microscopic diagnosis has gone through a paradigm shift with the development of new techniques such as the quantitative buffy coat (QBC) method and the Partec rapid malaria test. This review explores the different diagnostic tools available for childhood malaria, each with their characteristic strengths and limitations. These tools play an important role in making an accurate malaria diagnosis to ensure that the use of anti-malaria are rationalized and that presumptive diagnosis would only be a thing of the past.

Matched MeSH terms: Real-Time Polymerase Chain Reaction/methods
Fulltext Dietary Lecithin Decreases Skeletal Muscle COL1A1 and COL3A1 Gene Expression in Finisher Gilts

Akit H, Collins C, Fahri F, Hung A, D'Souza D, Leury B, et al.

Animals (Basel), 2016;6(6).
PMID: 27338483 DOI: 10.3390/ani6060038

The purpose of this study was to investigate the effect of dietary lecithin on skeletal muscle gene expression of collagen precursors and enzymes involved in collagen synthesis and degradation. Finisher gilts with an average start weight of 55.9 ± 2.22 kg were fed diets containing either 0, 4, 20 or 80 g/kg soybean lecithin prior to harvest for six weeks and the rectus abdominis muscle gene expression profile was analyzed by quantitative real-time PCR. Lecithin treatment down-regulated Type I (α1) procollagen (COL1A1) and Type III (α1) procollagen (COL3A1) mRNA expression ( p < 0.05, respectively), indicating a decrease in the precursors for collagen synthesis. The α-subunit of prolyl 4-hydroxylase (P4H) mRNA expression also tended to be down-regulated ( p = 0.056), indicating a decrease in collagen synthesis. Decreased matrix metalloproteinase-1 (MMP-1) mRNA expression may reflect a positive regulatory response to the reduced collagen synthesis in muscle from the pigs fed lecithin ( p = 0.035). Lecithin had no effect on tissue inhibitor metalloproteinase-1 (TIMP-1), matrix metalloproteinase-13 (MMP-13) and lysyl oxidase mRNA expression. In conclusion, lecithin down-regulated COL1A1 and COL3A1 as well as tended to down-regulate α-subunit P4H expression. However, determination of muscle collagen content and solubility are required to support the gene functions.

Matched MeSH terms: Real-Time Polymerase Chain Reaction
Fulltext Differential expression of basal microRNAs' patterns in human dental pulp stem cells

Vasanthan P, Govindasamy V, Gnanasegaran N, Kunasekaran W, Musa S, Abu Kasim NH

J Cell Mol Med, 2015 Mar;19(3):566-80.
PMID: 25475098 DOI: 10.1111/jcmm.12381

MicroRNAs (miRNAs) are small non-coding RNAs that regulate translation of mRNA into protein and play a crucial role for almost all biological activities. However, the identification of miRNAs from mesenchymal stem cells (MSCs), especially from dental pulp, is poorly understood. In this study, dental pulp stem cells (DPSCs) were characterized in terms of their proliferation and differentiation capacity. Furthermore, 104 known mature miRNAs were profiled by using real-time PCR. Notably, we observed 19 up-regulated miRNAs and 29 significantly down-regulated miRNAs in DPSCs in comparison with bone marrow MSCs (BM-MSCs). The 19 up-regulated miRNAs were subjected to ingenuity analysis, which were composed into 25 functional networks. We have chosen top 2 functional networks, which comprised 10 miRNA (hsa-miR-516a-3p, hsa-miR-125b-1-3p, hsa-miR-221-5p, hsa-miR-7, hsa-miR-584-5p, hsa-miR-190a, hsa-miR-106a-5p, hsa-mir-376a-5p, hsa-mir-377-5p and hsa-let-7f-2-3p). Prediction of target mRNAs and associated biological pathways regulated by each of this miRNA was carried out. We paid special attention to hsa-miR-516a-3p and hsa-miR-7-5p as these miRNAs were highly expressed upon validation with qRT-PCR analysis. We further proceeded with loss-of-function analysis with these miRNAs and we observed that hsa-miR-516a-3p knockdown induced a significant increase in the expression of WNT5A. Likewise, the knockdown of hsa-miR-7-5p increased the expression of EGFR. Nevertheless, further validation revealed the role of WNT5A as an indirect target of hsa-miR-516a-3p. These results provide new insights into the dynamic role of miRNA expression in DPSCs. In conclusion, using miRNA signatures in human as a prediction tool will enable us to elucidate the biological processes occurring in DPSCs.

Matched MeSH terms: Real-Time Polymerase Chain Reaction

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